The oxygen carrying portion of red blood cells is the protein hemoglobin. Hemoglobin is a tetrameric molecule composed of two identical alpha globin subunits (.alpha..sub.1, .alpha..sub.2), two identical beta globin subunits (.beta..sub.1, .beta..sub.2) and four heme molecules, with one heme incorporated per globin. Heme is a large macrocyclic organic molecule containing an iron atom; each heme can combine reversibly with one ligand molecule such as oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form a stable alpha/beta dimer, two of which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.
Severe blood loss often requires replacement of the volume of lost blood as well as the oxygen carrying capacity of that blood. This replacement is typically accomplished by transfusing red blood cells (RBC's), either as packed RBC's or as units of whole blood. However, it is not always possible, practical or desirable to transfuse a patient with donated blood. Human blood transfusions are associated with many risks such as, for example, transmission of diseases and disease causing agents such as human immunodeficiency virus (HIV), hepatitis, Yersinia enterocolitica, cytomegalovirus, and human T-cell leukemia virus. In addition, blood transfusions can be associated with immunologic reactions such as hemolytic transfusion reactions, immunosuppresion, and graft versus host reactions. Moreover, blood must be typed and cross-matched prior to administration, and may not be available due to limited supplies.
When human blood is not available or the risk of transfusion is too great, plasma expanders can be administered. However, plasma expanders, such as colloid and crystalloid solutions, replace only blood volume, and not oxygen carrying capacity. In situations where blood is not available for transfusion, a red blood cell substitute that can transport oxygen in addition to providing volume replacement is desirable. Solutions of cell-free hemoglobin can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, but, in addition, a hemoglobin-based red blood cell substitute can support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution can be used in most situations where red blood cells are currently utilized. For example, oxygen-transporting hemoglobin-based solutions can be used to temporarily augment oxygen delivery during or after pre-donation of autologous blood prior to the return of the autologous blood to the patient.
To address this need, a number of red blood cell substitutes have been developed (Winslow, R. M.(1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). These substitutes include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 0307087), stroma-free hemoglobin solutions, both chemically crosslinked and uncrosslinked, derived from a variety of mammalian red blood cells (Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Bos. 4,826,811 and 5,194,590; Vlahakes, G J. et al., (1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed in and purified from genetically engineered organisms (for example, non-erythrocyte cells such as bacteria and yeast, Hoffman et al., WO 90/13645; bacteria, Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood cell substitutes have been designed to replace or augment the volume and the oxygen carrying capability of red blood cells.
However, red blood cell replacement solutions that have been administered to animals and humans have exhibited certain adverse events upon administration. These adverse reactions have included hypertension, renal failure, neurotoxicity, and liver toxicity (Winslow, R. M., (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp.; Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. Biotech. 20: 1013-1020). In the case of perfluorocarbons, hypertension, activation of the reticulo-endothelial system, and complement activation have been observed (Reichelt, H. et al., (1992) in Blood Substitutes and Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. supra, pp. 778-781). For hemoglobin based oxygen carriers, renal failure and renal toxicity is the result of the formation of hemoglobin .alpha./.beta. dimers. The formation of dimers can be prevented by chemically crosslinking (Sehgal, et al., U.S. Pat. Nos. 4,826,811 and 5,194,590; Walder, J. A. U.S. Reissue Pat. No. RE34271) or genetically linking (Hoffman, et al., WO 90/13645) the hemoglobin dimers so that the tetramer is prevented from dissociating.
Prevention of dimer formation has not alleviated all of the adverse events associated with hemoglobin administration. Blood pressure changes and gastrointestinal effects upon administration of hemoglobin solutions have been attributed to vasoconstriction resulting from the binding of endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and constitutive NO are primarily produced in the endothelium of the vasculature and act as local modulators of vascular tone.
When hemoglobin is contained in red blood cells, it cannot move beyond the boundaries of blood vessels. Therefore, nitric oxide must diffuse to the hemoglobin in an RBC before it is bound. When hemoglobin is not contained within an RBC, such as is the case with hemoglobin-based blood substitutes, it may pass beyond the endothelium lining the blood vessels and penetrate to the extravascular space (extravasation). Thus, a possible mechanism causing adverse events associated with administration of extracellular hemoglobin may be excessive inactivation of nitric oxide due to hemoglobin extravasation. Furthermore, NO is constitutively synthesized by the vascular endothelium. Inactivation of NO in the endothelium and extravascular space may lead to vasoconstriction and the pressor response as well as other side effects observed after infusions of cell-free hemoglobin. Larger hemoglobins may serve to reduce hypertension associated with the use of some extracellular hemoglobin solutions.
In addition, the half-life of these molecules is limited and is much lower than hemoglobin that is contained within red blood cells. Such short-lived hemoglobin is accordingly rapidly cleared from the body and may not be appropriate for oxygen delivery over longer periods of time, from hours to days. Hemoglobin that is intramolecularly and/or intermolecularly crosslinked by a chemical crosslinker may have an increased halve. The increased half-life may be due to the inhibition of hemoglobin clearance mechanisms by the presence of the crosslinker in the three-dimensional structure of the hemoglobin. Such chemical crosslinkers may interfere with clearance processes such as haptoglobin binding or binding to other specific hemoglobin receptors.
As discussed above, hemoglobin from any source can be chemically crosslinked using a variety of chemistries. Aldehydes such as glutaraldehyde and glycolaldehyde have been used to crosslink hemoglobin both intramolecularly (within a tetramer) and intermolecularly (between tetramers). Intramolecular crosslinks serve to prevent dimerization into alpha/beta dimers and may also alter oxygen affinity and cooperativity, while intermolecular crosslinks create polymers of tetrameric hemoglobin. Polymeric hemoglobins may result in reduced extravasation because of their increased size. Reduced extravasation may, in turn, lead to reduced pressor effects resulting from infused hemoglobin solutions.
One hemoglobin tetramer binds four oxygen molecules. Because hemoglobin is a cooperative molecule, the binding of one oxygen molecule at one heme increases the ease with which the next oxygen molecule is bound. The combination of oxygen affinity and cooperativity of the hemoglobin molecule determines the ease with which the molecule binds and releases oxygen. Both contribute to the shape of the oxygen equilibrium binding curve, which in turn controls the binding of oxygen to hemoglobin in the lungs and the release of oxygen from hemoglobin in the tissues (Bunn and Forget, Hemoglobin: Molecular, Genetic and Clinical Aspects, (1986) W. B. Saunders, Philadelphia, Pa., pp 37-60). Therefore, either or both of these functionalities of the hemoglobin molecule can be adjusted to yield a hemoglobin that has suitable parameters for a given application. It is generally thought that an effective blood substitute should have moderately low oxygen affinity and should exhibit some level of cooperative binding of oxygen. Lower oxygen affinities and some preservation of cooperativity can be achieved if the hemoglobin is modified with chemicals designed to reduce oxygen affinity such as pyridoxal-5'-phosphate and related compounds (Snyder and Walder in Biotechnology of Blood, J. Goldstein, editor, Butterworth-Heinemann, Boston, (1991) 101-116; Benesch and Benesch (1981), Meth. Enzymol. 76: 147-159), or the hemoglobin is very low oxygen affinity prior to crosslinking (e.g. bovine hemoglobin). Treatment of hemoglobin with additional reagents is cumbersome and increases the cost of the product by increasing the material costs and increasing the number of production and purification steps. Cooperativity of the molecule is often significantly reduced during chemical treatments, and is difficult to maintain at levels found in the molecule prior to chemical treatment. Generally, it is desirable to produce a hemoglobin-based blood substitute with more cooperativity rather than less cooperativity.
For use in physiological applications, the hemoglobin should be intramolecularly crosslinked to avoid dimerization and concommittant renal toxicity. Crosslinking of hemoglobin with polyfunctional crosslinkers has been previously described (Bonsen et al., U.S. Pat. No. 4,053,590; Bonhard and Boysen, U.S. Pat. No. 4,336,248; Sehgal et al., U.S. Pat. No. 4,826,811; Hsia, U.S. Pat. No. 5,364,932, see Vandegriff, K. D.(1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. Tombs, Editor, Intercept Ltd., Andover, England, and Winslow, R. M.(1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp for reviews). However, crosslinking of the hemoglobin in these cases generally yields hemoglobins with higher oxygen affinity (lowered P.sub.50) and significantly reduced cooperativity (lower n or n.sub.max) than the hemoglobin that was used as starting material. Chemical crosslinking of hemoglobin, as practiced to date, provides a system to create stabilized tetramers or high molecular weight hemoglobins. However, it is not possible, using existing technologies, to reduce the significant loss of cooperativity of the hemoglobin molecule during chemical crosslinking. Thus, a need exists for methods of controlling loss of cooperativity of intra- or intermolecularly chemically crosslinked hemoglobins by methods that do not require the use of additional chemicals, for example, by regulating deoxygenation or protein concentration of the non-polymerized hemoglobin. The present invention satisfies this need and provides related advantages.